Haynes:TransfectionPlasmid Lipo

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(New page: <- Back to Protocols <div style="width: 800px" align="center"> '''Mammalian Cell Transfection - Lipofectamine Reagent'''<br> Karmella Haynes, 2012 </div> <div styl...)
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'''Mammalian Cell Transfection - Lipofectamine Reagent'''<br>
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'''Mammalian Cell Transfection - Lipofectamine LTX Reagent'''<br>
Karmella Haynes, 2012
Karmella Haynes, 2012
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<div style="width: 800px">
<div style="width: 800px">
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Note: Good for quick-and-dirty transfections; pretty toxic
 
'''Materials'''
'''Materials'''
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* Lipofectamine 2000
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* Lipofectamine LTX
* Opti-MEM I Reduced serum medium
* Opti-MEM I Reduced serum medium
* Antibiotic-free growth medium (no pen/strep or other antibiotics)
* Antibiotic-free growth medium (no pen/strep or other antibiotics)
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* Cells
'''6-well format'''<br>
'''6-well format'''<br>
'''1 day before transfection:'''<br>
'''1 day before transfection:'''<br>
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# Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection. Use antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep).
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# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well.
'''Transfection:'''<br>
'''Transfection:'''<br>
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# Warm plasmid DNA, Lipofectamine 2000, and Opti-MEM I Reduced Serum Medium to room temp.  
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# Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
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# Prepare DNA-Lipofectamine complexes (500 μl per well) as follows:
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# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
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## Label sterile microfuge ml tubes.
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## Label sterile microfuge (1.5 ml) tubes.
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## Dilute plasmid DNA (4 μg) in 250 μl of Opti-MEM in each tube.
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## For each transfection, bring '''2.0 μg plasmid DNA''' up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
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## Dilute 4.0 μl Lipofectamine 2000 in 250 μl of Opti-MEM (per sample). Incubate for 5 minutes at room temp. Do not let the mixture sit for more than 25 minutes.
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## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
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## Add 50 μl of the diluted Lipofectamine 2000 to the diluted DNA (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temp. Complexes are stable for 6 hours at room temp.
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## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
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## Add 500 μl of complexes to each well. Mix with gentle rocking.
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## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
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# Incubate cells at 37°C in a CO2 incubator for 5-6 hours. Wash cells once with warm 1xPBS. Add fresh antibiotic-free growth medium. Transgene expression should be detectable after 24 hours.
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# Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each well of cells.
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# Incubate cells at 37°C in a CO<sub>2</sub> incubator  
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# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.  
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Transgene expression should be detectable after 24 hours.
<br><br>
<br><br>
''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.
''For stable cells lines:'' Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.

Revision as of 18:34, 30 November 2012

<- Back to Protocols

Mammalian Cell Transfection - Lipofectamine LTX Reagent
Karmella Haynes, 2012

Materials

  • Lipofectamine LTX
  • Opti-MEM I Reduced serum medium
  • Antibiotic-free growth medium (no pen/strep or other antibiotics)
  • Cells

6-well format

1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection. Use 1.0 mL antibiotic-free growth medium (e.g., 10% FBS in McCoy’s 5A for, no pen/strep) per well.

Transfection:

  1. Warm plasmid DNA, Lipofectamine LTX, PLUS reagent, and Opti-MEM I Reduced Serum Medium to room temperature in the biosafety cabinet.
  2. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. For each transfection, bring 2.0 μg plasmid DNA up to 20 μL total volume with sterile DNase-free water. Take the DNA samples to the biosafety cabinet.
    3. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    4. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    5. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  3. Add DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each well of cells.
  4. Incubate cells at 37°C in a CO2 incubator
  5. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 24 hours.



For stable cells lines: Passage cells at a 1:10 (or higher) dilution into fresh growth medium 24 hours after transfection. Add selective medium the following day. Grow until isolated colonies appear. Transfer single colonies into 24-well plates. Expand the cells for further testing.
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